In the antisense technology, RNA is targeted by Watson-Crick hybridization of a complementary antisense oligonucleotide (AON). The goal of inhibiting gene expression in a specific way may be accomplished by preventing mRNA maturation, blocking translation or more commonly by induction of target RNA degradation [Crooke, S. T. Biochim. Biophys. Acta 1999, 1489, 31-44; Zamaratski, E.; Pradeepkumar, P. I.; Chattopadhyaya, J. J. Biochem. Biophys. Methods 2001, 48, 189-208]. To be effective the AON has to be able to enter the cell, be stable toward nucleases, be non-toxic and show high binding affinity and specificity toward the target mRNA. Considerable progress with respect to stability and binding has been made by use of chemically modified AONs. Introducing nucleotide analogues with constrained North type (N-type; C3′-endo type) furanose ring conformations has proven successful with respect to obtaining strong binding toward an RNA target, with LNA (locked nucleic acid) being a prominent example. An LNA monomer contains an O2′-C4′ methylene linkage that locks the furanose ring in an N-type conformation leading to unprecedented binding affinity toward complementary RNA for AONs composed of a mixture of e.g. LNA and DNA nucleotides [Koshkin, A. A.; Singh, S. K.; Nielsen, P.; Rajwanshi, V. K.; Kumar, R.; Meldgaard, M.; Olsen, C. E.; Wengel, J. Tetrahedron 1998, 54, 3607-3630; Obika, S.; Nanbu, D.; Hari, Y.; Andoh, J.-i.; Mono, K.-i.; Doi, T.; Imanishi, T. Tetrahedron Lett. 1998, 39, 5401-5404; Petersen, M.; Wengel, J. Trends Biotechnol. 2003, 21, 74-81; Vester, B.; Wengel, J. Biochemistry 2004, 43, 13233-13241].
Incorporation of LNA nucleotides into an AON induces formation of almost canonical A-form helix structures of the duplexes formed with RNA complements [Petersen, M.; Bondensgaard, K.; Wengel, J.; Jacobsen, J. P. J. Am. Chem. Soc. 2002, 124, 5974-5982; Nielsen, K. E.; Rasmussen, J.; Kumar, R.; Wengel, J.; Jacobsen, J. P.; Petersen, M. Bioconjugate Chem. 2004, 15, 449-457; Nielsen, C. B.; Singh, S. K.; Wengel, J.; Jacobsen, J. P. J. Biomol. Struct. Dyn. 1999, 17, 175-191; Nielsen, K. E.; Singh, S. K.; Wengel, J.; Jacobsen, J. P. Bioconjugate Chem. 2000, 11, 228-238], and LNA thus can be characterized as a structural mimic of RNA though it lacks the 2′-OH group of an RNA nucleotide. Contrary, the stereoisomeric α-L-LNA monomer is locked in a conformation that results in AONs that structurally mimic DNA whereby duplexes between DNA/α-L-LNA mixmers and RNA adopt intermediate A/B duplex geometries [Nielsen, K. M.; Petersen, M.; Hakansson, A. E.; Wengel, J.; Jacobsen, J. P. Chem. Eur. J. 2002, 8, 3001-3009; Petersen, M.; Håkansson, A. E.; Wengel, J.; Jacobsen, J. P. J. Am. Chem. Soc. 2001, 123, 7431-7432. Remarkably, both LNA and α-L-LNA nucleotides induce very high RNA binding affinities of AONs with increases in thermal denaturation temperatures (Tm values) of ˜2-8° C. per modification [Vester, B.; Wengel, J. Biochemistry 2004, 43, 13233-13241; Sørensen, M. D.; Kvaerno, L.; Bryld, T.; Hakansson, A. E.; Verbeure, B.; Gaubert, G.; Herdewijn, P.; Wengel, J. J. Am. Chem. Soc. 2002, 124, 2164-2176]. LNA-modified oligonucleotides have likewise shown promising properties with respect to targeting microRNAs, i.e. as so-called antiMiRs, and one LNA oligonucleotide targeting microRNA 122 is currently in clinical phase 2 studies as a drug to treat HCV infection [www.santaris.com]. Furthermore LNA-modified oligonucleotides have shown promise as splice-modulating compounds [B. Bestas et al., J. Clin. Investigation, 2014, 9, 4067], and also as so-called blockmirs which are single-stranded oligonucleotides which target the microRNA binding sites [www.mirrx.dk].
A number of other locked nucleotides, i.e. analogs of LNA, such as BNAs, carbocyclic-LNAs and CEt have been studied in the context of therapeutic oligonucleotides [Rahman, S. M. A et al., Chem. Lett. 2009, 38, 512][Zhou, C. and Chattopadhyaya, J., Curr. Opin. Drug Disc. Devel., 2009, 12, 2180][Seth, P. P. and Swayze, E. E., in Natural Products in Medicinal Chemistry, Ed. Hanessian, S, Wiley-VCH, Weinheim, 1st ed., 2014, 203-439].
The efficiency of gapmer antisense oligonucleotides containing modified nucleotides is often limited by their inability to induce degradation of target mRNA by the ubiquitous RNase H enzyme. Specifically, RNase H is incompatible with substrate duplexes with N-type nucleotides like LNA or O2′-alkylated-RNA nucleotides dispersed throughout the AON [Sørensen, M. D.; Kvaerno, L.; Bryld, T.; Hakansson, A. E.; Verbeure, B.; Gaubert, G.; Herdewijn, P.; Wengel, J. J. Am. Chem. Soc. 2002, 124, 2164-2176; Lima, W. F.; Nichols, J. G.; Wu, H.; Prakash, T. P.; Migawa, M. T.; Wyrzykiewicz, T. K.; Bhat, B.; Crooke, S. T. J. Biol. Chem. 2004, 279, 36317-36326]. Such O2′-alkylated-RNA nucleotides can for example be 2′-O-methyl-RNA nucleotides or 2′-O-methoxyethyl-RNA (2′-MOE-RNA) nucleotides.
Aptamers, which herein are defined as short single-stranded oligonucleotides, are alternative oligonucleotide constructs for drug development. Aptamers adopt well-defined three-dimensional shapes which enables targeting of for example peptides, proteins, small molecules, viruses and live cells [Eckstein, F., Expert Opin. Biol. Ther. 2007, 7, 1021; Famulok, M. et al., Chem. Rev. 2007, 107, 3715; Thiel, K. W. and Giangrande, P. H., Oligonucleotides 2009, 19, 209; Mayer, G. Angew. Chem. Int. Ed. 2009, 48, 2672]. In solution, the nucleotides together constituting the sequence of the aptamer have the potential to form segments of base-paired regions which will induce folding of the molecule into a complex three-dimensional shape thereby ideally allowing the aptamer to bind tightly against the surface of its target molecule. It is the capability of aptamers to form diverse molecular shapes depending on their sequence that enable them to form binding interactions with targets. Aptamers are typically generated by evolution of specific sequences against a given target by so-called in vitro evolution using the process known as SELEX (systematic evolution of ligands by exponential enrichment) [Ellington, A. D. and Szostak, J. W., Nature 1990, 346, 818; Turk, C. and Gold, L., Science 1990, 249, 5059]. SELEX involves iterative rounds of selection and polymerase-catalyzed enrichment (PCR) of bound aptamers selected from a pool of nucleic acid components, i.e. from a large library of typically e.g. 50-100 nucleotide long sequences involving a central variable (sequence randomized) region flanked by two primer-binding regions.
Aptamers are versatile drug candidates as they can be evolved against extracellular targets like receptors or certain signaling molecules. They can also be evolved against intracellular components and thereby mediate a biological response once internalized into cells. Aptamers have been applied in vivo to specifically deliver an anti-cancer siRNA to prostate cancer cells [Dassie, J. P. et al., Nature Biotechnology 2009, 27, 839]. Intracellular application of aptamers is a challenging task due to poor intracellular delivery caused by their relatively large size and polyanionic character. Another challenge is biodistribution as some unconjugated oligonucleotides are excreted rapidly via the kidneys upon i.v. administration. PEGylation (chemical attachment by so-called conjugation of polyethylene glycol), and lipid nanoparticulate formulation are example approaches which have been applied to improve biodistribution [Veronese, F. M. et al., Drug Disc. Today 2005, 10, 1451].
One aptamer has been approved as drug (pegaptanib; to treat age-related macular degeneration upon local administration in the eye) and others are or have been in various stages of clinical development towards different diseases [Famulok, M., J. Med. Chem. 2009, 52, 6951]. These therapeutic candidates have generally been obtained by so-called post-SELEX modification of aptamers which have been evolved by a full SELEX procedure. Post-SELEX modification typically involves truncation into shorter aptamer candidates, conjugation (e.g. pegylation) for improved biodistribution, and/or incorporation of chemically modified nucleotides for improved biostability. Post-SELEX chemical modification is necessary as only rather few modified nucleoside triphosphates, for example 2′-fluoro-RNA, 2′-amino-RNA and 5-substituted pyrimidine nucleoside triphosphates [Mayer, G., Angew. Chem. Int. Ed. 2009, 48, 2672], are substrates for the polymerase-catalyzed reactions required for efficient SELEX procedures. Post-SELEX modifications are typically performed in iterative rounds of synthesis and binding assays/biological evaluation to ensure that modifications are compatible with the desired aptamer properties.
AS1411 is a 26-mer G-rich oligodeoxynucleotide. Like the vast majority of aptamers its nucleoside constituents are linked together by natural phosphodiester (PO) linkages as these are highly compatible with the SELEX procedure. AS1411 has been reported to fold as a stable dimeric G-quadruplex structure [Collie G. W. and Parkinson, G. N. Chem. Soc. Rev. 2011, 40, 5867]. It can cause induction of cell death in human cancer cell lines and has little effect on normal cells, and it has been tested in phase I and II clinical trials of patients with advanced cancer [Reyes-Reyes, E. M. et al., Cancer Res. 2010, 70, 8617]. Its target has been identified as nucleolin though its mechanism of action is not completely understood. It has been proposed that nucleolin-binding leads to selective uptake of AS-1411 into cancer cells. More recent studies have confirmed the involvement of nucleolin in the action of AS1411 and have suggested that uptake of AS1411 may be by macropinocytosis [Reyes-Reyes, E. M. et al., Cancer Res. 2010, 70, 8617].
Some derivatives of and properties of acyl-amino-LNA and hydrocarbyl (such as alkyl)-amino-LNA monomers and oligomers are known from published scientific papers. A review on amino-LNA derivatives—including acyl-amino-LNA and alkyl-amino-LNA [I. K. Astakhova and J. Wengel, Acc. Chem. Res., 2014, 47, 1768] has recently been published. When compared to the corresponding DNA/RNA oligonucleotides, acyl-amino-LNA and hydrocarbyl (such as alkyl)-amino-LNA oligonucleotides generally display increased affinity towards complementary DNA/RNA strands much in line with the increased affinity observed for the corresponding LNA oligonucleotides.
Glycyl-amino-LNA and palmitoyl-amino-LNA monomers can be mixed with LNA and DNA monomers [Johannsen, M. W. et al., Org. Biomol. Chem., 2011, 9, 243]. Nucleotide monomers composed of pyrene linked to amino-LNA monomers via an N2′-linker have been reported to be useful as fluorescent probes, and acyl-amino-LNA derivatives containing various amino acids as acyl group have been shown to be compatible (mixable in the same strand or oligonucleotide) with DNA nucleotides, and 2′-amino-LNA and 2′-N-methyl-amino LNA monomers have been shown to be useful in gapmer antisense oligonucleotides [see I. K. Astakhova and J. Wengel, Acc. Chem. Res., 2014, 47, 1768 and references cited therein].
It has been reported that oligonucleotides containing a piperazino-modified 2′-amino-LNA monomer exhibit high duplex stability and remarkable nuclease resistance. The nuclease resistance was for the studied oligonucleotide (mixmer with DNA, phosphodiester (PO) linkages) even significantly increased compared to the corresponding oligonucleotide containing a parent amino-LNA monomer instead of the piperazino-modified 2′-amino-LNA monomer [Lou, C., Vester, B. and Wengel, J. Chem. Comm. 2015. 19, 4024-4027]. And the induced nucleolytic stability was shown to be entended towards the 3′-end of the oligonucleotide several DNA nucleotides away from the piperazino-modified 2′-amino-LNA monomer. These results demonstrate that the oligonucleotides of the invention, even as all-PO oligonucleotide, display significant stabilization against nucleolytic degradation.
No reports have been published on the use of acyl-amino-LNA, e.g. glycyl- or palmitoyl-amino-LNA-containing oligonucleotides for RNA targeting in vivo, e.g. in the context of antisense, antimir or blockmir, or as compounds able to modulate splicing events.
The invention discloses the use of oligonucleotides containing two or more acyl-amino-LNA and/or hydrocarbyl-amino-LNA monomers as RNA-targeting constructs for therapeutic or diagnostic purposes. Examples of the invented constructs include gapmer antisense constructs, mixmer antisense constructs, antimir constructs, blockmir constructs and aptamer constructs.
The use of gapmer antisense constructs, mixmer antisense constructs, antimir constructs, blockmir constructs and aptamer constructs containing 2′-amino-LNA (“2′-NH”) and 2′-N-methyl-amino-LNA (“2′-NCH3”) monomers as only amino-LNA type monomer are not included in the present invention.
The invention further discloses the use of oligonucleotides containing two or more acyl-amino-LNA and/or hydrocarbyl-amino-LNA monomers as nucleic acid aptamers as non-RNA-targeting constructs for therapeutic or diagnostic purposes.
The oligonucleotides of the invention can be used to mediate RNA targeting in organs of animals or humans not reached effectively by standard single-stranded RNA-targeting oligonucleotides, e.g. phosphorothioate-LNA oligonucleotides or phosphorothioate-MOE oligonucleotides. Such effect is mediated because a wide range of hydrocarbyl and acyl groups can be attached to the N2′-position of amino-LNA monomers in hydrocarbyl-amino-LNA and acyl-amino-LNA monomers and incorporated into oligonucleotides. Thereby modulation of the pharmacokinetic properties of the oligonucleotide can be realized. For example, hydrophobic acyl or hydrocarbyl (such as alkyl) groups ease permeation across cell membranes and may furthermore lead to improved accumulation in liver tissues. Furthermore, many fatty acid residues like palmitoyl or myristoyl, when attached to the N2′-position of amino-LNA monomers in an oligonucleotide of the invention, lead to binding to plasma proteins, e.g. albumin, which in turn may lead to improved circulation time in the blood and improved tissue distribution and uptake.
The presence of two of such fatty acid conjugated amino-LNA modifications (e.g. palmitoyl-amino-LNA residues) is, in the invention, one particularly preferred design in order to achieve improved circulation time in the blood and improved tissue distribution and uptake for phosphodiester or phosphorothioate based oligonucleotides, e.g. gapmers or mixmers for targeting mRNA or non-coding RNAs—e.g. for miRNA targeting, modulation of splice switching, miRNA target site blockage, gene silencing or upregulation of gene expression. In the case of phosphodiester variants of such antisense oligonucleotides, the stability against nucleolytic degradation is increased compared to the corresponding oligonucleotides composed e.g. as LNA-DNA-LNA or BNA-DNA-BNA gapmers or as LNA/DNA, BNA/DNA, LNA/2′-O-Me-RNA, BNA/2′-O-Me-RNA or LNA/DNA/2′-OMe-RNA mixmers, in particular when two of the LNA or BNA monomers are exchanged by the acyl- or amino-LNA monomers of the invention.
Also acyl or hydrocarbyl (such as alkyl) groups carrying one or more positive charge(s) can be beneficial for cell membrane permeability and tissue targeting. Other options include conjugation with carbohydrates like galactose units or CPPs (cell penetrating peptides). The presence of at least two acyl-amino-LNA and/or hydrocarbyl (such as alkyl)-amino-LNA monomer in an oligonucleotide improves the nuclease stability relative to unmodified or LNA-containing reference oligonucleotides. The fact that groups of different sizes can be attached without disturbing the RNA-targeting capability enables engineering of desired biostability e.g. engineering of increased stability against degradation by nucleases.
For antisense applications, so-called gapmers are often used. These are chimeric AONs with a central continuous stretch of RNase H recruiting nucleotides (typically DNA or phosphorothioate DNA nucleotides but alternatively e.g. phosphorothioate FANA nucleotides [Lok, C. N.; Viazovkina, E.; Min, K. L.; Nagy, E.; Wilds, C. J.; Damha, M. J.; Parniak, M. A. Biochemistry 2002, 41, 3457-3467]) flanked by affinity-enhancing modified nucleotides (e.g. LNA, α-L-LNA or O2′-alkylated RNA nucleotides) [Jepsen, J. S.; Sørensen, M. D.; Wengel, J. Oligonucleotides 2004, 14, 130-146; Monia, B. P.; Lesnik, E. A.; Gonzalez, C.; Lima, W. F.; McGee, D.; Guinosso, C. J.; Kawasaki, A. M.; Cook, P. D.; Freier, S. M. J. Biol. Chem. 1993, 268, 14514-14522; Kurreck, J.; Wyszko, E.; Gillen, C.; Erdmann, V. A. Nucleic Acids Res. 2002, 30, 1911-1918; Frieden, M.; Christensen, S. M.; Mikkelsen, N. D.; Rosenbohm, C.; Thrue, C. A.; Westergaard, M.; Hansen, H. F.; Orum, H.; Koch, T. Nucleic Acids Res. 2003, 31, 6365-6372]. It has been found that the optimal gap size is motif-dependent, that a right balance between gap size and affinity is required [Kurreck, J.; Wyszko, E.; Gillen, C.; Erdmann, V. A. Nucleic Acids Res. 2002, 30, 1911-1918], and that the presence of one or two DNA-mimicking α-L-LNA monomers within the gap is compatible, at least in part, with RNase H activity [Sørensen, M. D.; Kvaerno, L.; Bryld, T.; Hakansson, A. E.; Verbeure, B.; Gaubert, G.; Herdewijn, P.; Wengel, J. J. Am. Chem. Soc. 2002, 124, 2164-2176; Frieden, M.; Christensen, S. M.; Mikkelsen, N. D.; Rosenbohm, C.; Thrue, C. A.; Westergaard, M.; Hansen, H. F.; Orum, H.; Koch, T. Nucleic Acids Res. 2003, 31, 6365-6372].
Acyl- and hydrocarbyl (such as alkyl)-amino-LNA monomers have previously been incorporated into DNA strands, and therefore procedures for preparation of their phosphoramidite building blocks for automated oligonucleotide synthesis have been reported as well as procedures for their incorporation into oligonucleotides (e.g. DNA, RNA, LNA, UNA (unlocked nucleic acids) or 2′-OMe-RNA oligonucleotides) [see I. K. Astakhova and J. Wengel, Acc. Chem. Res., 2014, 47, 1768 and references cited therein, in particular Johannsen, M. W. et al., Org. Biomol. Chem., 2011, 9, 243].
The following structural and functional features are considered optimal for antisense type oligonucleotides (such as gapmers or mixmers) by the inventor:                (a) relatively small size, i.e. below 20 nucleotides in total;        (b) containing phosphodiester (PO), or a majority of PO, internucleoside linkages instead of PS internucleoside linkages;        (c) a long half-life in serum;        (d) limited excretion via the kidneys (urine);        (e) stability against nucleolytic degradation;        (f) ability to penetrate cell membranes and mediate modulation of gene expression without the addition of transfecting agents or the use of nanoparticulate formulations.        
Fulfilment of a) is advantageous for production purposes, and may further aid cell membrane permeation and/or biodistribution. Incorporation of LNA-type nucleotides (or BNA-type nucleotides) is the preferred way of reducing the length of a therapeutic oligonucleotide because of the high RNA target affinity induced by LNA (or BNA) nucleotides [Obad, S. et al. Nature Genetics 2011, 43, 371-378]. Incorporation of LNA (or BNA) nucleotides may also enable the development of short therapeutic aptamers as the excellent hybridization properties of LNA (or BNA) nucleotides may promote intramolecular structuring even for shorter aptamer sequences.
Fulfilment of b) is advantageous in order to avoid the formation of diastereoisomeric mixtures generated at each PS (phosphorothioate) linkage. A 16-mer all-PS standard antisense oligonucleotide as an example, may exist as a mixture of 215 (=32.768) distinct molecules [Wan, W. B. et al., Nucleic Acids Res. 2014, 42, 13456-13468], of which some may contribute to the desired therapeutic action while others may give rise, for example, to undesired effects. Some off-target effects of oligonucleotides containing PS linkages may originate from non-specific binding to proteins and immune-related side effects [Jastrzebska et al. Org. Biomol. Chem. 2015, 13, 10032-10040 and references cited therein]. These challenges of standard PS-oligonucleotides has prompted research into synthesis of P-stereodefined PS oligonucleotides, but preparation of such compounds requires the use and development of cumbersome synthetic methods and have not furnished improved therapeutic derivatives [Wan, W. B. et al., Nucleic Acids Res. 2014, 42, 13456-13468; Jastrzebska et al. Org. Biomol. Chem. 2015, 13, 10032-10040]. Therapeutic oligonucleotides containing preferentially, or exclusively, PO linkages are considered desired as they would show less off-target effects.
Fulfilment of c), d) and e) is desirable to obtain the pharmacokinetic properties needed for oligonucleotide drug development. To fulfil these three points, PS linkages have typically been used to achieve binding to plasma proteins thus reducing excretion via the kidneys, and furthermore protection against degradation by nucleases. However, still a significant amount of undesired and rapid excretion via the kidneys is observed. Alternatively lipid derivatives, most typically cholesteryl, have been applied to mediate binding to plasma proteins. This has in particular been studied for siRNA constructs (double stranded RNAs) [Wolfrum, C. et al., Nature Biotech. 2007, 25, 1149-1157; Howard, K. A., Bienk, K. and Kragh-Hansen, U. WO2014/005596], but still no lipid derivative of an siRNA or a single stranded oligonucleotide has been approved as a drug, and the challenges prevail [Wittrup, A. and Lieberman, J., Nature Rev. Gen. 2015, 16, 543-552]. Interestingly, promising gene silencing activity using transfection agent and albumin was reported for singly or doubly cholesteryl-modified siRNAs, but notably not for the corresponding C12-C16 fatty acid functionalized siRNAs [Wolfrum, C. et al., Nature Biotech. 2007, 25, 1149-1157; Howard, K. A., Bienk, K. and Kragh-Hansen, U. WO2014/005596].
For aptamers, fulfilment of c) and d) has typically been achieved by conjugation with PEG (polyethylene glycol) units [Hirota, M. et al, Nucleic Acids Ther. 2016, 26, 10-19]. This however is a non-preferred solution. Firstly, conjugation with PEG is an additional complication towards obtaining the final drug component, and secondly have immune reactions against PEG units and rapid clearance of PEGylated systems been reported [Yang, Q. and Lai, S. K., Advanced Review 2015, 7, 655-677].
It has been reported that efficient so-called gymnotic delivery (or unassisted delivery with no transfection agent used) and concomitant gene modulation activity of an oligonucleotide correlates particularly well with in vivo activity, and such efficient unassisted permeation into cells is therefore now considered highly important for oligonucleotide drug development efforts. Efficient unassisted delivery has been demonstrated for high-affinity LNA-type antisense gapmers [Stein, C. A. et al. Nucleic Acids Res. 2010, 38, e3; Zhang, Y. et al. Gene Therapy 2011, 18, 326-333] and for the relatively high-affinity 2′-FANA oligonucleotides [Souleimanian, N. et al., Mol. Ther. Nucleic Acids 2012, 1, e43]. Fulfilment of f) has thus been achieved by using oligonucleotides containing high-affinity monomers, but notably only in the context of PS linkages. It should be mentioned that the IC50 values obtained for inhibition of gene expression typically have been in the low micromolar range, significantly above the nanomolar range often obtained when using transfection agents. Further it should be noted that standard all-PO oligonucleotides have failed to display activity or uptake under unassisted delivery conditions.
Based on the single stranded oligonucleotides reported so far in the literature, no construct has fulfilled all six desirable points a)-f) listed and discussed above. The following summarizes some key points based on reported data:
1) All-PS oligonucleotides display improved pharmacokinetic properties relative to all-PO oligonucleotides but their therapeutic use is hampered by undesirable off-target effects and a relatively high rate of renal clearance;
2) Double cholesteryl-functionalization of siRNA constructs have been shown to be compatible with efficient gene silencing in vitro using transfection-/permeation-aiding molecules, while double palmitoyl-functionalization of the same siRNAs failed to demonstrate efficient gene silencing under similar conditions;
3) Improved circulatory half-life, i.e. reduced renal clearance rate, has been reported for cholesteryl and palmitoyl functionalized siRNA;
4) Stability against exo- and endonuclease for single stranded oligonucleotides has typically required the use of chemically modified nucleotides, either at most of the nucleotide positions in the strand, or with the use of PS linkages in substantially all linkage positions.
5) Single stranded oligonucleotides containing more than 30-40% RNA nucleotides and PO linkages are typically unstable towards nucleolytic degradation.
6) Efficient cell uptake and gene silencing activity under unassisted delivery conditions requires the use of single stranded oligonucleotides having PS linkages in most of the linkage positions.
It is an object of the present invention to provide single-stranded oligonucleotides having high transfection efficiency in eukaryotic cells or cells of an organism such as an animal or a human, even if no transfectants are used.
It is an object of the present invention to provide oligonucleotides that have long half-life in serum of an animal such as a mammal.
It is an object of the present invention to provide antisense oligonucleotides which, when bound to RNA target sequences, are efficient substrates of RNase H type enzymes.
It is another object of the invention to provide antisense oligonucleotides which, without being a substrate of RNase H, bind strongly to target RNA, thereby leading to modulation of gene expression. The target RNA can be mRNA or non-coding RNAs.
It is another object of the invention to provide antisense oligonucleotides with improved properties with regard to stability towards enzymatic degradation in cell cultures or in vivo. Still another object is to provide antisense oligonucleotides that display improved bioavailability, increased tissue distribution or otherwise improved properties, e.g. improved gene silencing effect in vivo, relative to the corresponding antisense oligonucleotides not containing two or more acyl-amino-LNA and/or hydrocarbyl-amino-LNA monomers.
It has surprisingly been found by the inventor that the single stranded oligonucleotides of the invention containing two or more acyl-amino-LNA or hydrocarbyl (such as alkyl)-amino-LNA nucleotide monomers fulfil all desirable points a)-f) listed above. The oligonucleotides of the invention thus display the following:    7) they are efficient as relatively short strands because of the LNA-type high-affinity hybridization mediated by the acyl-amino-LNA and hydrocarbyl (such as alkyl)-amino-LNA nucleotide monomers;    8) they are efficiently taken up by cells and display gene modulation activity under unassisted delivery conditions, and that even as all-PO oligonucleotides (i.e. having PO at all linkages or at most of the linkages), but also as all-PS derivatives:    9) they display increased half-life in serum as all-PO and all-PS derivatives, even when compared to the corresponding all-PS oligonucleotides without the acyl-amino-LNA and hydrocarbyl-amino-LNA nucleotide monomers;    10) they show less accumulation in the kidneys as all-PO and all-PS derivatives, even when compared to the corresponding all-PS oligonucleotides without the acyl-amino-LNA and alkyl-amino-LNA nucleotide monomers;    11) they show satisfactory stability against nucleolytic degradation as also testified by their substantial serum half-life, and that both as all-PO and all-PS derivatives;    12) they display the ability to penetrate cell membranes and mediate modulation of gene expression without the need of addition of transfecting agents or the use of nanoparticulate formulations, and that both as all-PO and all-PS derivatives.